Structure-guided and high-throughput engineering of genetically encoded sensors for reactive oxygen species

活性氧基因编码传感器的结构引导和高通量工程

基本信息

批准号：
10337219
负责人：
Andre Berndt
金额：
$ 40.22万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10337219
关键词：
Acute Affect Alzheimer&apos s Disease Amyloid beta-Protein Biophysics Cardiac Myocytes Cardiomyopathies Cardiovascular Diseases Cell physiology Cells Cellular Metabolic Process Chronic Color Coupling DNA Damage Detection Directed Molecular Evolution Disease Disease Progression Disease model Drug Screening Dyes Engineering Environment Failure Feedback Fluorescent Probes Functional disorder Future Gene Expression Genetic Genetic Engineering Goals Green Fluorescent Proteins Human Hydrogen Peroxide Hypoxia Impairment Individual Intervention Ischemia Kinetics Libraries Link Lipids Location Measurement Measures Methods Microscopy Mitochondria Modeling Monitor Mutation Nerve Degeneration Neurons Organ Oxidative Stress Oxygen Pathologic Patients Performance Pharmacology Phenotype Physiologic Monitoring Physiological Physiological Processes Preparation Problem Solving Process Protein Engineering Proteins Randomized Reactive Oxygen Species Reperfusion Injury Reperfusion Therapy Repression Research Personnel Resolution Rhodamine Role Signal Transduction Site Specificity Speed Stress Structure Technology Tertiary Protein Structure Testing Time Variant base biophysical properties cell type cellular targeting design detection sensitivity disease phenotype high throughput screening improved in vivo induced pluripotent stem cell innovation novel novel therapeutic intervention polydimethylsiloxane protein expression public health relevance receptor red fluorescent protein relating to nervous system response restraint screening sensor stem cell model stem cells stressor subcellular targeting tool

项目摘要

Modified Project Summary/Abstract Section Elevated levels of reactive oxygen species (ROS) are strongly linked to severe pathological conditions causing cardiomyopathies and neurodegeneration. Today we can utilize fluorescent probes to detect dynamic changes in ROS levels in cell physiology and pathophysiology. However, the capabilities of many ROS sensors are currently still limited by small signal amplitudes, slow kinetics, low sensitivity, in vivo incompatibility, and restraints in cellular and subcellular targeting. Thus, monitoring ROS of oxidative stress in real-time is still very restricted. Our central goal in this proposal is to resolve current limitations in ROS protein sensors. We will combine structured-guided protein design and high-throughput screening of large variant libraries in an innovative approach to engineer novel ROS sensors. We expect that significantly increasing signal amplitudes, ROS sensitivity, and insensitivity to hypoxic conditions will enable us to monitor oxidative stress in a wide range of disease models. Furthermore, we will validate new sensors in stem-cell derived models for neurodegeneration and cardiomyopathies with subcellular precision. Our objective is to further maximize ROS sensor function for advanced monitoring of oxidative stress in disease models in response to acute and chronic stressors. In the first aim, we will broaden the color spectrum of this class of sensors by fusing green, yellow, and red fluorescent proteins to a ROS sensitive protein domain . Furthermore, we will create sensors that are more photostable and insensitive to varying oxygen levels compared to fluorescent proteins. In the second aim, we will use a novel engineering platform for fluorescent sensors that allows us to screen large libraries of randomized variants. The fast, iterative process has the potential to significantly accelerate the optimization of sensor frameworks established in Aim 1. In the third aim, we will validate our sensors in several realistic use scenarios to receive immediate feedback for further refinement of sensor function. This includes the monitoring of oxidative stress as an indicator for Alzheimer’s disease, ischemia and reperfusion in stem-cell-derived neurons and cardiomyocytes. This proposal is significant because oxidative stress is common and can affect every organ and cell type resulting in a large number of severe diseases. Recent progress in fluorescent microscopy allows us to utilize specific probes to monitoring physiological processes with increasing precision. The engineering of improved ROS sensors will significantly expand the utility of those methods for the analysis of cell signaling and disease progression. Our project is innovative because the proposed approach will provide the fastest throughput for the design of highly efficient ROS sensor proteins. Furthermore, the improved sensors will be able to causally link disease phenotypes to acute and chronic stressors of oxidative stress with significantly increased temporal and spatial resolution.

修改的项目摘要/摘要部分活性氧（ROS）的水平升高与导致心肌病和神经变性的严重病理状况密切相关。今天，我们可以利用荧光问题来检测细胞生理和病理生理学中ROS水平的动态变化。但是，许多ROS传感器的功能目前受到小信号放大器的限制，缓慢的动力学，低灵敏度，体内不相容性以及细胞和亚细胞靶向的约束。那是实时监测氧化应激的ROS仍然非常限制。我们在此提案中的核心目标是解决ROS蛋白传感器中的当前局限性。我们将结合结构化引导的蛋白质设计和对大型变体库的高通量筛选，以一种创新的方法来设计新颖的ROS传感器。我们期望明显增加信号放大器，ROS敏感性和对低氧条件的不敏感性，这将使我们能够在广泛的疾病模型中监测氧化应激。此外，我们将验证具有亚细胞精度的神经变性和心肌病的干细胞衍生模型中的新传感器。我们的目标是进一步最大化ROS传感器功能，以响应急性和慢性应激，以高级监测疾病模型中氧化应激的监测。在第一个目标中，我们将通过将绿色，黄色和红色荧光蛋白融合到ROS敏感蛋白结构域来扩展此类传感器的色谱。此外，与荧光蛋白相比，我们将创建对氧气水平变化的传感器。在第二个目标中，我们将使用一个新颖的工程平台来用于荧光传感器，使我们能够筛选大型随机变体库。快速的迭代过程有可能显着加速AIM 1中建立的传感器框架的优化。在第三个目标中，我们将在几种现实的使用场景中验证传感器，以接收即时反馈，以进一步完善传感器功能。这包括监测氧化应激，作为阿尔茨海默氏病，缺血和再灌注的指标，在干细胞衍生的神经元和心肌细胞中。该建议很重要，因为氧化应激很常见，并且会影响大量严重疾病的每种器官和细胞类型。荧光显微镜的最新进展使我们能够利用特定的问题来监测精度。改进的ROS传感器的工程将显着扩大这些方法的效用，以分析细胞信号传导和疾病进展。我们的项目具有创新性，因为所提出的方法将为高效ROS传感器蛋白的设计提供最快的吞吐量。此外，改进的传感器将能够偶尔将疾病表型与氧化应激的急性和慢性应激联系起来，并显着增加临时和空间分辨率。